Most water-borne human pathogens cause infections and human disease via ingestion of fecal contaminated water or food. Various human parasites and pathogens are transmitted in this way, including protozoa, virus and bacteria, transmitted via human fecal contamination of water used for drinking, bathing, recreation, harvesting of shellfish, or washing/preparation of foods. Additionally, some water-borne pathogens are transmitted via contaminated aerosols and enter the human body through the respiratory tract. Legionella pneumophila, the causative agent of legionellosis fatal respiratory pneumonia infection know as Legionnaire's Disease, is transmitted in this manner. Warm stationary domestic water found in air conditioner cooling towers, inadequately chlorinated swimming pools and spas, hot water heaters, respiratory therapy equipment and shower heads, have been identified as sources of Legionella infectious outbreaks. The need for and adequacy of water purification and the safety of natural (ground and surface) waters for recreation, drinking and shellfish harvest, routinely is monitored by standard microbiological tests for fecal flora and for Legionella. The control of legionellosis requires environmental monitoring so that the reservoirs of L. pneumophila can be identified. Decontamination procedures can then be implemented in order to reduce or eliminate this bacterial pathogen, thereby reducing the risk of outbreaks of legionellosis. Additionally, the sources of L. pneumophila must be quickly determined when outbreaks of legionellosis occur to prevent additional cases.
Because many fecal pathogens are hard to assay or are infective at densities so low that water sample collection and concentration is inconvenient, fecal microbial water contamination often is assessed by testing for harder and more robust, but not necessarily pathogenic, microbes, referred to as indicator organisms, such as the "fecal coliforms", especially Escherichia coli.
The most common tests for both fecal bacteria indicator organisms and Legionella require culturing for one to several days on/or in a nutrient medium under standard conditions, followed by counting of the colonies which develop or identification of positive liquid cultures and the use of a most probably number table to determine the numbers of bacteria in the sample. Various metabolic, biochemical and immunochemical tests may be used to confirm the microbiological identities of the organisms enumerated in these tests, although they require additional time, expense and skilled labor.
Culture tests of microbiological contamination are tedious, time consuming, unsatisfactorily slow, delaying decisions which might seriously affect human health and which test should be made on the time scale of hours, not days.
There are several problems with viable culture methods used for routine monitoring of the bacteriological safety of water supplies, including the tediousness of the method, maintaining viability of bacteria between the time of collection and enumeration, lack of growth of viable but nonculturable bacteria--such as those stressed by chemicals in the water, failure to cultivate all living cells of interest, time (days) required for detection and confirmation of enteric bacteria, lack of specificity for detection of true fecal coliforms such as E. Coli, failure to distinguish living from dead cells using direct microscopic counts, and misidentification of organisms due to antigenic cross reactivity using serological procedures.
Usually such culture tests are not as specific or as sensitive as desirable to ensure with sufficient certainty the presence or absence of specific microbes in low numbers. Gene probes that specifically hybridize with the DNA of specific regions associated with specific pathogens or indicator organisms provides a means of specific detection but conventional gene probe methods are generally about 10,000 times less sensitive than required for environmental monitoring purposes.
Recently, tests based upon detection of .beta.-D-glucuronidase, such as the Colilert test, have been suggested as alternate approaches for detecting coliforms. Enzymatic transformation of the fluorogenic substrate 4-methylunbelliferyl-.beta.-glucuronidide or colorimetric substrate p-nitrophenyl glucuronide is indicative of the presence of E. coli, but detection of such enzymatic activity still requires culturing of bacteria. It has also recently been reported that basing a test on .beta.-D-glucuronidase activity may fail to detect a significant proportion, about 30%, of fecal coliform bacteria in some cases because of occurrences of high incidences of .beta.-D-glucuronidase negative E. coli.
The traditional methods for the detection of L. pneumophila are the viable plate count and the direct microscopic count, the latter by using several commercially available fluorescent antibody reagents. Although cultivation of viable cells of Legionella from water samples is a standard procedure, it is tedious and time consuming. Additionally, cultivation methods may underestimate numbers of viable cells because some cells of L. pneumophila are sensitive to the acid-wash treatment and selective media used in the standard viable enumeration procedure. Also, some cells may be viable but nonculturable. The serological detection of Legionella using polyclonal antibody reagents likewise has limitations because immunofluorescence microscopy cannot distinguish living from dead cells and also some non-Legionella cells show false positive reactions due to antigenic-cross reactivity.
Detection of Legionella has been reported by colony hybridization using a radiolabelled unique DNA gene probe fragment isolated from Legionella chromosomal DNA after restriction enzyme digestion. In waters with high microbial populations, as are found in water cooling towers, the level of detection sensitivity by colony hybridization would be about 10.sup.4 /ml. A sensitivity of 5.times.10.sup.4 cells was achieved by using a separate gene probe. A commercial gene probe detection kit, based upon rRNA detection, has a sensitivity of 10.sup.3 or 10.sup.4 cells.
It is therefore highly desirable that a process and kits therefor be available for detection of water-borne microbial pathogens and indicators of human fecal contamination in natural environmental, purified or other water sources in which the aforementioned problems are eliminated or substantially reduced. A further object of the invention is to provide such a process and kits which have greater specificity and sensitivity to ensure detection of the pathogens or indicators at very low concentrations. A still further object of this invention is to provide such a process and kits therefor which enable the noncultural detection of such pathogens and indicators. Another object of this invention is to provide such process and kits therefor which detect nonculturable coliforms. It is also an object of this invention to provide such a process and kits therefor which enable the detection of such pathogens and indicators in a matter of hours rather than days. It is a still further object of this invention to provide a process and kits therefor which permit direct detection of pathogens in water samples, rather than relying upon the indirect detection of indicator organisms.
An additional object of this invention is to provide an effective, specific and sensitive process and kits therefor for detection of sources of Legionella so as to prevent or determine the sources of outbreaks of legionellosis. A still further additional object of this invention is to provide such a process and kits therefor for specific detection of L. pneumophila for both clinical diagnosis and environmental monitoring or source tracing of L. pneumophila. It is another object of this invention to provide such a process an kits therefor in which one can detect both all Legionella species and all L. pneumophila serotypes in the same water sample at the same time.
An additional object of this invention is to provide a process and kits therefor which will detect substantially all species and strains which fall in the microbial pathogens or indicator class and no organisms from other species or genera. A still further object is to provide such a process and kits therefor which employ a highly polymorphic sequence within the target gene to permit distinction among variants and in some other cases to avoid any distinction among variants of the same target species, genus or other defined group.